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Mycobacterium Leprae a Nd Elevation Of Lepr. Rev. (1978) 49, 203-213 Absence of jj-Glucuron idase in Mycobacterium leprae and Elevation of the Enzyme in I nfected Tissues K. PRABHAKARAN, E. B. HARRIS AND W. F. KIRCHHEIMER U. S. Public Health Service Hospital. Carville. LA 70 721. USA ,8-Glucuronidase actlVlty was determined in mouse footpads infected with My cobacterium leprae. in the leprosy organisms separated from the liver and spleen of experimentally infected armadillos, and in the armadillo tissues. Enzyme assays in th� mouse footpads were initiated 1 week after inoculation with M. /eprae and continued at monthly intervals for 12 months. In the mouse footpads and in the armadillo tissues, M. leprae infection resulted in remarkable elevations of ,8- glucuronidase leveis. The leprosy bacilli seemed to be devoid of the enzyme. In its properties like pH optimum, reaction velocity and effect of inhibitors, the activity detected in M. leprae resembled the host tis sue enzyme rather than bacterial ,8- glucuronidase; and the activity was found to be superficially adsorbed on the bacilli. lt is well established that phagocytes are rich in lysosomal enzymes. Evidently, the increased ,8-g1ucuronidase of the infected tissues is not derived from the invading organisms, but from the differenttypes of phagocytic cells infiltratingthe tissues. Introduction j3-Glucuronidase is an important hydrolytic enzyme ubiquitously distributed in animal tissues and in tissue fluids. Phagocytic cells are especially rich in j3-g1ucuronidase. In the mammalian liver, the enzyme is largely associated with Iysosomes, and approximately one-third of the activity is distributed in the endoplasmic reticulum. The hydrolase is closely correlated with cellular pro­ liferation and tissue repair; high leveis of the enzyme are found in the reproductive and endocrine organs and in tumours. Skin cancers as well as normal skin contain j3-g1ucuronidase. The enzyme has been reported in molluscs and in insects. Among bacteria, Escherichia co li, Streptococcus pyogenes, Corynebacterium xerose, C. hofJmani and rumen micro-organisms have been shown to produce j3-g1ucuronidase (Fishman, 1955; Levy and Marsh, 1959). In bacterial infections characterized by intracellular parasitism of the invading organisms, marked e1evation of hydrolytic enzymes has been observed (AlIen, 1969). j3-Glucuronidase splits offD- glucuronic acid residues from oligosaccharides, thus degrading these complex molecules. In the body, glucuronic acid forms Received for publication 9 February 1978. 0305-75 18/78/090 1-0203$01 .00/0 IC> 1978 British Leprosy Relief Association 204 K. PRABHAKARAN ET AL. conjugates of hormones, drugs and other substances, thus serving a detoxifica­ tion function; also ,B-glucuronidase can hydrolyze such conjugates and release the organic compounds (Fishman, 1955). Because of their degradative activities, Iysosomal enzymes, including ,B-glucuronidase, have a major role in the killing and digestion of ingested bacteria (Allen, 1969). In leprosy, as irt certain other mycobacterial infections, a paradoxical situation occurs; the bacteria, instead of being destroyed by the phagocytes, seem to be protected in these cells (Goren, 1977; Brown et aI., 1969). No significant elevation of ,B-glucuronidase was observed in peripheral blood leucocytes of leprosy patients (Avila and Convit, 1970; Garcia­ Gonzalez et ai., 1977). Increase in acid phosphatase was demonstrated histochemically in histiocytes containing My cobacterium leprae (Brieger and Allen, 1962). Based on histochemical evidence, it was concluded that M. leprae possesses ,B-glucuronidase (Matsuo and Skinsnes, 1974). In the course of our investigations over the past several years on Iysosomal enzymes in leprosy, we studied the leveis of acid phosphatase and ,B-glucuronidase in mouse footpads and in tis sues of armadillos experimentally infected with M. leprae. We also determined ,B-glucuronidase activity in concentrates of the bacilli separated fr om infected armadillo tis sues. Because acid phosphatase is extremely labile, its assay was found to be unre\iable in tissues stored for varying periods of time. As such, acid phosphatase assays were not pursued fu rther. However, ,B-glucuronidase gave consistent and reproducible results. The data presented in this report show that M. leprae purified fr om infected armadillo tissues apparently does not contain ,B-glucuronidase; in the mouse fo otpad, the enzyme activity continues to rise as the infection progresses; and in the infected armadillo tis sues, the enzyme leveis are elevated 2 to 3 times over that of the uninfected tissues. Materiais and Methods ANIMALS ANO MYCOBACTERIA Approximately 8-week-old female Swiss mice ofthe NIH strain were used in the study. The experimental animais were inoculated in the left hind footpads with 1 x 104 M. leprae. The M. leprae used was a mouse-passage strain originally obtained fr om the skin biopsy of an untreated lepromatous patient. Control mice were of the same age and sexo Starting at 1 week after inoculation, a fe w mice (usually 5) were sacrificed at monthly intervals for 12 months. The soft tissues from the left hind footpads of both the experimental and the control animals were collected and stored at -80°C. At the time of assay, the material was thawed out, washed in cold saline, blotted and minced. The tissues were extracted in small amounts of water by grinding in a chilled agate mortar. The extract was centrifuged briefly, to remove the particulate niatter. Enzyme determinations were made in the supernatant fr action. Protein was estimated by the method of Lowry et aI. (195 1). Enumeration of M. leprae in the mouse footpad was done at 6 months and 12 months, by the method of Hanks et ai. (1964), modified by Kirchheimer. Liver and spleen of "normal" and infected armadillos were coUected GLUCURONIDASE AND LEPROSY 205 aseptically at autopsy and were held at O°C or at -80°C before processing. Ten percent homogenates of these organs were made in water. Suspensions of the leprosy bacilli were prepared as reported before (Prabhakaran et aI., 1976) from the liver or spleen of 9-banded armadillos experimentally infected with M. leprae (Kirchheimer and Storrs, 1971; Kirchheimer et ai., 1972). The bacilli were disrupted by ultrasonic oscillation (Prabhakaran et aI., 1973). In an effort to remove superficially adsorbed host-tissue materiais from the bacterial concentrate, it was treated with trypsin (final concentration 0.5%), N aOH (0. 1 N), sodium dodecyl sulfate (1%), or sodium deoxycholate (0.5%) for 30 min at o°C, except for trypsin, which was done at 37°C . My cobacterium phlei was grown in Proskauer-Beck medium (Youman's modification) at 37°C for 2 weeks. The bacilli were harvested by centrifuga­ tion and washed twice with cold saline and once with deionized glass-distilled water. ENZYMES AND CHEMICALS Purified E. coZi ,lj-glucuronidase (Type VII), bovine liver ,lj-glucuronidase (Type B-IO), phenolphthalein glucuronic acid (sodium salt) and glycine buffer were purchased fr om the Sigma Chemical Co., St Louis, MO, USA. Other chemicals used were ofthe highest purity commercially available. ENZYME ASSAY The enzyme assay was done essentially by the standard procedure of Talalay et aI. (1946). The reaction mixture consisted of the following con­ stituents: 0. 1 M acetate buffer (pH 4.7), 0.4 ml; 0.01 M phenolphthalein glu­ curonic acid, 0. 1 ml; sample, 0.5 ml. The sample blank contained 0.5 ml buffer and 0.5 ml sample, and the reagent blank 0.9 ml buffer and 0.1 ml substrate. For E. coZi ,lj-glucuronidase, 0.075 M phosphate buffer (pH 6.8) was substituted for acetate buffer; 40 units of the enzyme were added. The mycobacterial suspensions used contained 2-5 x 109 organisms/ml. The final concentration of the inhibitors tested was 0.005 M. After thereaction mixture was incubated at 37°C for 60 min, 1 ml of 5% TCA was added, followed by 2.5 ml of alkaline glycine reagent. Volume was made up to 6 ml with 1.5 ml of water. The reaction mixture was centrifuged and the purple colour of the supernatant fr action was read at 540 nm in a Beckman DU-2 spectrophotometer. The readings were corrected for any absorbance due to the sample or reagent blanks. The amount of phenolphthalein liberated from the substrate by the enzyme was calculated from a standard curve. All reactions were carried out in duplicate and each experiment was done at least 3 times. The results reported are mean values of representative experiments. Results MOUSE FOOTPADS M. leprae inoculated in the mouse footpads multiplied normally; enumeration of the bacilli showed 2.4 ± 0.18 x 106 organisms per footpad in 6 206 K. PRABHAKARAN ET AL. 60 50 O i;;c e o- O> 40 E ........ c � o .c 30 1:: Cl. o c ., .c 20 o- O' ::L 10 O 2 4 6 8 10 12 Time (months) Fig. I. ,B-glucuronidase of mouse footpads infected with M. leprae. (Á-Á) Experimental; (e-e) control. months, and 1.8±O.15x 107 in 12 months. j3-Glucuronidase actlVlty was assayed in the footpad tissues at monthly intervals for 12 months. To correct for variations in individual assays, the values obtained for both the control and the experimental samples were subjected to regression analysis. The results are presented in Fig. 1. In the uninfected mice, the enzyme activity remained fairly stable throughout the experimento However, j3-glucuronidase continued to rise in the footpads of mice where M. leprae multiplied progressively. These results do not indicate whether the higher activity is derived from the bacilli or from the cells that infiltrate the infected footpads. ARMADILLO TISSUES j3-Glucuronidase was determined in the liver and the spleen tissues of both the infected and the uninfected armadillos. (On the average, the infected organs TABLE 1 ,B-Glucuronidase in "normal" and irifected sp leen tissue of armadi/los: Ilg of phenolphthalein released/mg protein Number of animals "Normal" Infected 1 32.2 92.1 2 37.7 173.5 3 44.4 110.5 Mean 38.1 125.4 GLUCURONIDASE ANO LEPROSY 207 TABLE 2 f3-Glucuronidase in "normal" and infected liver tissue of armadillos: I1g of phenolphthalein releasedlmg protein N umber of animais "Normal" Infected 1 77.4 188.9 2 162.0 200.0 3 118.4 286.3 4 130.8 234.4 Mean 122.2 227.4 contained over 109 M.
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